Recombinant Artibeus fuliginosus Cytochrome b (MT-CYB)

What are the optimal storage conditions for recombinant MT-CYB protein?

For optimal stability and activity, recombinant MT-CYB should be stored according to the following protocol:

• Upon receipt, briefly centrifuge the vial to bring contents to the bottom

• For long-term storage, store at -20°C/-80°C in aliquots with 5-50% glycerol (50% glycerol is the standard recommended concentration)

• Avoid repeated freeze-thaw cycles as this significantly reduces protein stability

• Working aliquots can be stored at 4°C for up to one week

• Lyophilized powder has a shelf life of approximately 12 months at -20°C/-80°C, while liquid forms maintain stability for about 6 months

How should recombinant MT-CYB be reconstituted for experimental use?

The recommended reconstitution protocol is as follows:

• Centrifuge the vial briefly before opening to bring the contents to the bottom

• Reconstitute the protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage (50% is recommended as default)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• For storage buffer considerations, typical formulations use Tris/PBS-based buffer with 6% Trehalose at pH 8.0

What expression systems are used to produce recombinant MT-CYB, and how does this affect protein properties?

Recombinant Artibeus fuliginosus Cytochrome b is typically expressed in E. coli expression systems. The protein is usually tagged with a His tag at the N-terminus to facilitate purification. The expression methodology impacts several protein characteristics:

• Purity: Expression in E. coli systems typically yields proteins with >85-90% purity as determined by SDS-PAGE

• Folding: While E. coli can express the protein efficiently, post-translational modifications might differ from the native bat protein

• Functionality: The recombinant protein retains its structural integrity, but activity assays should validate functional properties

• Tag influence: The His tag facilitates purification but may marginally affect certain protein-protein interactions in some experimental setups

How is MT-CYB used in phylogenetic studies of bat species, and what are the methodological considerations?

The cytochrome b gene is widely used in bat phylogenetics and taxonomic studies due to its evolutionary properties. Key methodological considerations include:

• Primer Design and Amplification Strategy:

  • For most bat species, standard primers (like DW1/DW6) successfully amplify the cytochrome b gene

  • For challenging genera like Eptesicus, custom reverse primers may be necessary

  • Nested PCR approaches using primers DW2/DW4 for initial amplification followed by DW1/DW6 for nested reactions yield approximately 1.1 kb fragments (~92% of the gene)

• Sequence Analysis and Phylogenetic Reconstruction:

  • Alignment should be performed using established algorithms like ClustalW

  • The GTR+G+I model typically best describes the substitution pattern based on Bayesian Information Criterion scores

  • Bayesian inference methods with Markov Chain Monte Carlo searches (8 million generations) provide robust phylogenetic reconstructions

  • Standard deviation of split frequencies should be <0.01 for reliable results

• Resolution Limitations:

  • Cytochrome b shows heterogeneous taxonomic resolution across bat genera

  • It provides species-level delimitation in non-conflicting genera (Eumops, Dasypterus, Molossops)

  • Only infrageneric resolution is possible in taxonomically challenging lineages (Eptesicus, Myotis, Molossus)

  • For Artibeus species, cytochrome b effectively resolves phylogenetic relationships with clear genetic distance patterns

Studies show that Artibeus fuliginosus MT-CYB maintains consistent genetic patterns across various populations, with genetic distance estimates between Artibeus species ranging from 3.6% to 14.0%, as detailed in Table 1:

Table 1: Genetic Distance Estimates Between Artibeus Species

These metrics form the basis for reliable species identification and evolutionary relationship analysis .

What role does MT-CYB play in mitochondrial-nuclear communication, and how can this be studied experimentally?

Recent research has revealed MT-CYB's unexpected role in nuclear gene regulation through mitochondrial-nuclear retrograde signaling. This communication pathway contributes to cellular homeostasis and stress responses. Experimental approaches to study this interaction include:

• Single Molecule Fluorescence In Situ Hybridization (smFISH):

  • Design smFISH probes specific to MT-CYB that don't cross-react with nuclear genome sequences

  • Use RNAscope with double Z probes to ensure specificity

  • Co-stain with nuclear markers (DAPI) and mitochondrial markers (Mitotracker)

  • Include Rho0 cells (lacking mitochondrial DNA) as negative controls

  • Use super-resolution imaging to capture nuclear localization of MT-CYB signals

• Sequential smFISH with Co-immunofluorescence:

  • Combine MT-CYB smFISH with immunofluorescence using SC35 antibody to mark nuclear speckles and active transcription regions

  • Design intron-targeting probes for pre-mRNAs of interest to track transcriptional effects

  • Three-dimensional reconstruction of confocal images to visualize MT-CYB signals embedded in DAPI-stained nuclei

• Functional Validation:

  • Compare normal vs. stressed conditions (e.g., hyperthermia treatment of endothelial cells)

  • Quantify nuclear localization of MT-CYB under different conditions

  • Analyze proximity of MT-CYB signals to induced pre-mRNAs

These approaches have revealed that MT-CYB RNA can localize to the nucleus as distinct puncta, particularly under stress conditions, suggesting a direct role in nuclear transcriptional regulation.

How can researchers design experiments to study the effect of MT-CYB mutations on respiratory chain function?

Studying the impact of MT-CYB mutations on respiratory chain function requires a multi-faceted approach:

• Molecular Dynamics Simulation:

  • Generate protein coordinates using I-TASSER or similar tools

  • Run simulations using NAMD program with CHARMM22 force field

  • Simulate in water-dissolved protein environment for 10-20 nsec under spherical boundary conditions

  • Maintain rigid water H-bonds using langevin dynamics at a constant temperature of 310 K

  • Analyze trajectories with VMD 1.9 and generate Ramachandran plots

  • Determine protein secondary structure changes using STRIDE

• Yeast Model Systems:

  • Introduce equivalent mutations in yeast cytochrome b using homologous recombination

  • Assess enzyme activity through spectrophotometric assays

  • Measure steady-state levels of bc1 complex using Blue-Native PAGE (BN-PAGE)

  • Evaluate respiratory growth on non-fermentable carbon sources

• Human Cell Studies:

  • For detection of MT-CYB mutations in patient samples:

    • Amplify the gene using PCR with appropriate primers

    • Sequence using Big Dye Terminator Cycle Sequencing Reaction Kits

    • For heteroplasmy quantification, design mismatched primers creating restriction sites for endonucleases (e.g., BanII)

    • Run digested fragments on 15% non-denaturing polyacrylamide gels and quantify using SYBR Gold Nucleic Acid Stain

What techniques are most effective for studying the assembly of MT-CYB into the respiratory chain complex III?

Investigating MT-CYB incorporation into complex III requires specialized techniques that monitor protein-protein interactions and complex assembly:

• Blue-Native PAGE (BN-PAGE):

  • This non-denaturing electrophoretic technique preserves protein-protein interactions

  • Sample preparation requires gentle solubilization of mitochondrial membranes with mild detergents (digitonin or n-dodecyl-β-D-maltoside)

  • Resolved complexes can be further analyzed by:

    • In-gel activity assays using specific substrates

    • Second-dimension SDS-PAGE to separate individual subunits

    • Western blotting with antibodies against complex III subunits

• Complexome Profiling:

  • A powerful technique that combines BN-PAGE with mass spectrometry

  • Enables identification of assembly intermediates and subcomplexes

  • Can detect aberrant early-stage subassemblies in mutants

  • Particularly useful for studying MT-CYB C-terminal region's role in complex assembly

• Assembly-Feedback Regulation Analysis:

  • Studies of yeast strains have revealed that absence of certain complex III subunits (e.g., Qcr7) reduces Cytb synthesis through an assembly-feedback mechanism

  • Key components of this mechanism include:

    • Cbp3 and Cbp6 proteins that assist Cytb synthesis

    • Cbp4 protein that induces Cytb hemylation

    • Qcr7/Qcr8 subunits that participate in early assembly steps

  • The C-terminal region of Cytb is critical for this regulation

Research has demonstrated that the C-terminal region of MT-CYB is essential for both regulation of synthesis and complete assembly of the bc1 complex. Deletion of this region results in loss of assembly-feedback regulation, allowing normal MT-CYB synthesis even when key assembly factors are missing, but prevents formation of a functional complex III .

How can MT-CYB be used in bat coronavirus host-pathogen interaction studies?

Recombinant MT-CYB can serve as a valuable tool in bat coronavirus research, particularly in understanding host-pathogen interactions. Key experimental approaches include:

• SARS-CoV-2 Challenge Models:

  • Artibeus species (like A. jamaicensis, related to A. fuliginosus) show distinct infection patterns compared to other bats

  • When challenged with SARS-CoV-2, infection is primarily confined to the intestine with viral nucleocapsid antigen in epithelial cells and mononuclear cells

  • Expression levels of ACE2 (the viral receptor) are low in bat lungs, which may account for limited pulmonary infection

  • Recombinant MT-CYB can be used as a control protein in these studies to evaluate mitochondrial function during infection

• Immune Response Analysis:

  • Bats exhibit distinct T cell responses to coronavirus infection

  • CD4+ helper T cells from infected bats show activation upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptides

  • These T cells express elevated mRNA levels of regulatory cytokines (IL-10, TGF-β)

  • MT-CYB can serve as a reference protein to normalize expression levels in these studies

• Evolutionary Adaptation Studies:

  • MT-CYB sequences from different bat species can be compared to identify potential correlations between mitochondrial function and viral susceptibility

  • This approach can help understand how evolutionary adaptations in energy metabolism might influence host-pathogen interactions

These approaches provide insights into how bats can harbor coronaviruses without developing clinical disease, potentially informing therapeutic strategies for human coronavirus infections.

What methodologies are available for studying the role of MT-CYB in haemosporidian parasite infections in bats?

Haemosporidian parasites, including Polychromophilus species that are exclusive to bats, can be studied using MT-CYB as a molecular marker. Comprehensive methodological approaches include:

• Molecular Detection of Haemosporidian Parasites:

  • PCR amplification of the mitochondrial cytb gene from parasite DNA:

    • Nested PCR approach using primers DW2/DW4 for first reaction

    • DW1/DW6 primers for nested reaction yielding ~1.1 kb fragment

    • Additional sequencing with DW8 and DW3 oligonucleotides

  • For enhanced detection, multi-gene approaches using:

    • Cytochrome b (cytb, 725 bp)

    • Adenylosuccinate lyase gene (asl, 206 bp)

    • Caseinolytic protease C gene (clpc, 531 bp)

• Phylogenetic Analysis:

  • Alignment using ClustalW algorithm implemented in MEGAX

  • Model selection: GTR+G+I typically best describes substitution patterns

  • Bayesian inference execution:

    • Two Markov Chain Monte Carlo searches (8 million generations)

    • Sampling 1 of 300 trees

    • After 25% burn-in, remaining trees used to calculate 50% majority-rule consensus tree

    • Standard deviation of split frequencies should be <0.01

• Field Sampling Strategies:

  • Target remaining fragments of natural habitats (e.g., Atlantic Forest, Pantanal biomes) and urbanized areas

  • Sample multiple bat species for comparative analysis

  • Include controls in all PCR amplifications:

    • Positive control (DNA sample with known Polychromophilus infection)

    • Negative control (ultrapure water) to check for contamination

These approaches have revealed that Polychromophilus parasites show genetic proximity to P. murinus in Brazilian Myotis bats, and have identified novel Haemosporida parasite sequences in species like Noctilio albiventris that show phylogenetic proximity to avian Haemoproteus sequences .